Esters of 19-hydroxy prostaglandin b2

ABSTRACT

A GROUP OF PROSTAGLANDINS, 19-HYDROXY-PGA2, 19-HYDROXY PGA2, 19-HYDROXY-PGB1, AND 19-HYDROXY-PGB2, AND THEIR SALTS, ESTERS, AND ACYLATES, ARE DISCLOSED. THESE ARE USEFUL FOR A VARIETY OF PHARMACOLOGICAL USES, INCLUDING BLOOD PRESSURE LOWERING, TREATMENT OF ASTHMA AND ULCERS, RELIEF OF NASAL CONGESTION, AND WOUND HEALING.

1973 B. SAMUELSSON ESTERS OF lQHYDROXY PROSTAGLANDIN B Filed June 21,1972 2.50 3552 0 1 2953:, 002 52 Ill 2253:. xooz 2 3 5 81.2 m amveaosavUnited States Patent O US. Cl. 260-468 D 10 Claims ABSTRACT OF THEDISCLOSURE A group of prostaglandins, l9-hydroxy-PGA 19-hydroxy PGA19-hydroXy-PGB and 19-hydroxy-PGB and their salts, esters, and acylates,are disclosed. These are useful for a variety of pharmacological uses,including blood pressure lowering, treatment of asthma and ulcers,relief of nasal congestion, and wound healing.

CROSS REFERENCE TO RELATED APPLICATIONS This application is acontinuation-in-part of my pending application Ser. No. 17,830, filedMar. 9, 1970, now abandoned, which is a continuation-in-part of mycopending application Ser. No. 481,777, filed Aug. 23, 1965, and nowabandoned.

DESCRIPTION OF THE INVENTION This invention relates to novelcompositions of matter, and to methods for obtaining, producing, andusing them. In particular, this invention relates to certain derivativesof prostanoic acid which has the following structural formula andnumbering:

The two chains in Formula I are attached to the cyclopentane ring at C-8and C-12 are in trans configuration. See Bergstrom et al., J. Biol.Chem. 238, 3555-3564 (1963) and Horton, Experientia 21, 113 (1965).

The compounds of this invention include essentially pure compounds ofthe formulas:

on (n O W t coon,

" ice In Formulas II, III, IV, and V, R is hydrogen or apharmacologically acceptable cation.

The compounds of this invention also include alkyl esters and alkanoatesof the formulas:

o CQOR, C"

Won. (v1) H H on; v11

0 ooom mom vm W C 000R,

In Formulas VI, VII, VHI, and IX, R is hydrogen, alkyl of one to 8carbon atoms, inclusive, or a pharmacologically acceptable cation, and Ris hydrogen or alkanoyl of one to 8 carbon atoms, inclusive, providedthat when R is hydrogen, R is alkyl of one to 8 carbon atoms, inclusive.

Molecules of the compounds encompassed by Formulas II to IX each haveseveral centers of asymmetry. Each of these 'Formulas II to IX isintended to represent optically active compounds each with the sameabsolute configuration as optically active prostaglandin E (PGE laternamed prostaglandin E (PGE and obtained from certain mammalian tissues,for example, sheep vesicular glands. See US. Pat. No. 3,069,322. Seealso later publications, for example, Bergstrom et al., J. Biol. Chem.238, 3555 (1963), Bergstrom et al., Pharmacol. Rev. 20, 1 (1968),I-Iamberg et al., J. Biol. Chem. 241, 257 (1966), Hamberg et al., NobelSymposium 2, Prostaglandins, Ed. S. Bergstrom and B. 'Samuelsson, pp.6370, Interscience Publishers, New York, 1967, Hamberg, European I.Biochem. 6, 147 (1968), and references cited in those.

In Formulas II to IX, a broken line attachment to the cyclopentene ringindicates a chain in alpha configuration, i.e., below the plane of thecyclopenteue ring. A heavy solid line attachment to the cyclopentenering indicates a chain in beta configuration, i.e., above the plane ofthe cyclopentane ring. The configuration of the side chain hydroxy atC-lS is alpha or S. The configuration of the side chain hydroxy at C-l9is beta or R.

With regard to Formulas VI, VII, VIII, and IX, examples of alkyl of oneto 8 carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, and isomeric forms thereof.

Examples of alkanoyl of one to 8 carbon atoms, inclusive, are formyl,acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, andisomeric forms thereof.

Pharmacologically acceptable cations within the scope of R in FormulasII, III, IV, and V, and within the scope of R in Formulas VI, VII, VIII,and IX are pharmacologically acceptable metal cations, ammonium ion,cations derived from pharmacologically acceptable amines, andpharmacologically acceptable quaternary ammonium cations.

Especially preferred metal cations are those derived from the alkalimetals, e.g., lithium, sodium and potassium, and from the alkaline earthmetals, e.g., magnesium and calcium, although cationic forms of othermetals, e.g., aluminum, zinc, and iron, are within the scope of thisinvention.

Pharmacologically acceptable amine cations are those derived fromprimary, secondary, or tertiary amines. Examples of suitable amines aremethylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine,triisopropylamine, N-methylhexylamine, decylamine, dodecylamine,allylamine, crotylamine, cyclopentylamine, dicyclohexylamine,benzylamine, dibenzylamine, a-phenylethylamine, fi-phenylethylamine,ethylenediamine; diethylenetriamine, and like aliphatic, cycloaliphatic,and araliphatic amines containing up to and including about 18 carbonatoms, as well as heterocyclic amines, e.g., piperidine, morpholine,pyrrolidine, piperazine, and loweralkyl derivatives thereof, e.g.,l-methylpiperidine, 4-ethylmorpholine, l-isopropylpyrrolidine,Z-methylpyrrolidine, 1,4- dimethylpiperazine, Z-methylpiperidine, andthe like, as well as amines containing water-solubilizing or hydrophilicgroups, e.g., mono-, di-, and triethanolamine, ethyldiethanolamine,N-butylethanolamine, 2 amino-l-butanol, 2-amino-2-ethyl-1,3-propanediol,2-amino-2-methyl-1-propanol, tris(hydroxymethyl)aminomethane,N-phenylethanolamine, N-(p-tertamylphenyl)diethanolamine, galactamine,N-methylglucamine, N-methylglucosamine, ephedrine, phenylephrine,epinephrine, procaine, and the like.

Examples of suitable pharmacologically acceptable quaternary ammoniumcations are tetramethylammonium, tetraethylammonium,benzyltrimethylammonium, phenyltriethylammonium, and the like.

Prostanoic acid derivatives of Formulas II to IX are valuabletherapeutic agents for the treatment of hypertension, in normalizingserum lipids and thus reducing the danger of ischaemic heart disease,and in treating central nervous system disorders in mammals includingman. These compounds are administered by intravenous infusion of sterileisotonic saline solutions at the rate of about 0.01 to about 10,preferably, about 0.1 to about 0.2, microgram per kilogram of animalweight per minute. These compounds are also useful because th y can beadministered to laboratory animals, preferably rats, to produce animalscontaining high levels of the compounds. Such animals can then serve astest animals in the search for and study of compounds which areantagonists of the administered compounds and which, for that reason,would be useful in reversing the effects of inadvertent overdoses of theextremely potent Formula II to IX compounds and in the treatment ofallergic conditions. For that purpose, the Formula II to IX compound isadvantageously administered to the test animal by continuous intravenousinfusion in sterile saline solution at the rate of about 0.01 to about10, preferably 0.05 to 0.2, microgram per kilogram of animal weight perminute until the desired level of compound has been reached or until thedesired response in the animal has been obtained. Infusion can then becontinued or stopped depending on the particular use to be made of thetest animal.

The compounds of Formulas II to IX and especially the PGA-type compoundsof Formulas II, III, VI and VII, are useful in mammals, including man,as nasal decongestants. For this purpose, the compounds are used in adose range of about 10 g. to about 10 mg. per ml. of a pharmacologicallysuitable liquid vehicle or as an aerosol spray, both for topicalapplication.

The PGA-type compounds of Formulas II, III, VI, and VII are useful inthe treatment of asthma. For example, these compounds are useful asbronchodilators or as inhibitors of mediators, such as SRS-A, andhistamine which are released from cells activated by an antigenantibodycomplex. Thus, these compounds control spasm and facilitate breathing inconditions such as bronchial asthma, bronchitis, bronchiectasis,pneumonia and emphysema. For these purposes, these compounds areadministered in a variety of dosage forms, e.g., orally in the form oftablets, capsules, or liquids; rectally in the form of suppositories;parenterally, subcutaneously, or intramuscularly, with intravenousadministration being preferred in emergency situations; by inhalation inthe form of aerosols or solutions for nebulizers; or by insufiiation inthe form of powder. Doses in the range of about 0.1 to 5 mg. per kg. ofbody weight are used 1 to 4 times a day, the exact dose depending on theage, weight, and condition of the patient and on the frequency and routeof administration. For the above use these prostaglandins can becombined advantageously with other anti-asthmatic agents, such assympathornimetics (isoproterenol, phenylephrine, ephedrine, etc);xanthine derivatives (theophylline and aminophyllin); andcorticosteroids (ACTH and predinisolone) The PGA-type compounds ofFormulas II, III, VI, and VII are also useful in mammals, including manand certain useful animals, e.g., dogs and pigs, to reduce and controlexcessive gastric secretion, thereby reducing or avoidinggastrointestinal ulcer formation, and accelerating the healing of suchulcers already present in the gastrointestinal tract. For this purpose,the compounds are injected or infused intravenously, subcutaneously, orintramuscularly in an infusion dose range about 1 g. to about 2 mg. perkg. of body weight per minute, or in a total daily dose by injection orinfusion in the range about 1 to about 50 mg. per kg. of body weight perday, the exact dose depending on the age, weight, and condition of thepatient or animal, and on the frequency and route of administration.

The PGA-type compounds of Formulas II, III, VI, and VII increase theflow of blood in the mammalian kidney, thereby increasing volume andelectrolyte content of the urine. For that reason, the PGA-typecompounds are useful in managing cases of renal disfunction, especiallyin cases of severely impaired renal blood flow, for example, thehepatorenal syndrome and early kidney transplant rejection. In cases ofexcessive or inappropriate ADI-I (antidiuretic hormone; vasopressin)secretion, the diuretic eifect of these compounds is even greater. Inauephric states, the vasopressin action of these compounds is especiallyuseful. Illustratively, the PGA-type compounds are useful to alleviateand correct cases of edema resulting, for example, from massive surfaceburns, and in the management of shock. For these purposes, the PGA-typecompounds are preferably first administered by intravenous injection ata dose in the range 100 to 10,000 #g. per kg. of body Weight or byintravenous infusion at a dose in the range 1 to 200 [Lgper kg. of bodyweight per minute until the desired eifect is obtained. Subsequent dosesare given by intravenous, intramuscular, or subcutaneous injection orinfusion in the range 0.5 to 5 mg. per kg. of body weight per day.

The PGB-type compounds of Formulas IV, V, VIII, and IX promote andaccelerate the growth of epidermal cells and keratin in animals,including humans, useful domestic animals, pets, zoological specimens,and laboratory animals. For that reason, these compounds are useful topromote and accelerate healing of skin which has been damaged, forexample, by burns, wounds, and abrasions, and after surgery. Thesecompounds are also useful to promote and accelerate adherence and growthof skin autografts, especially small, deep (Davis) grafts which areintended to cover skinless areas by subsequent outward growth ratherthan initially, and to retard rejection of homografts.

For these purposes, these compounds are preferably administeredtopically at or near the site where cell growth and keratin formation isdesired, advantageously as an aerosol liquid or micronized powder spray,as an isotonic aqueous solution in the case of wet dressings, or as alotion, cream, or ointment in combination with the usualpharmaceutically acceptable diluents. In some instances, for example,when there is substantial fluid loss as in the case of extensive burnsor skin loss due to other causes, systemic administration isadvantageous, for example, by intravenous injection or infusion,separate or in combination with the usual infusions of blood, plasma, orsubstitutes thereof. Alternative routes of administration aresubcutaneous or intramuscular near the site, oral, sublingual, buccal,rectal, or vaginal. The exact dose depends on such factors as the routeof administration, and the age, weight, and condition of the subject. Toillustrate, a wet dressing for topical application to second and/orthird degree burns of skin area 5 to 25 square centimeters wouldadvantageously involve use of an isotonic aqueous solution containing 1to 500 [.Lg-/III1. of the PGB-type compound. Especially for topical use,these prostaglandins are useful in combination with antibiotics, forexample, gentamycin, neomycin, polymyxin B, bacitracin, spectinomycin,and oxytetracycline, with other antibacterials, for example, mafenidehydrochloride, sulfadiazine, furazolium chloride, and nitrofurazone, andwith corticoid steroids, for example, hydrocortisone, prednisolone,methylprednisolone, and fluprednisolone, each of those being used in thecombination at the usual concentration suitable for its use alone.

The compounds of Formulas II to IX are used for the purposes describedabove in free acid form, in ester form, in dialkanoate form, inester-dialkanoate form, or in pharmacologically acceptable salt form.When the alkyl ester form is used, preferred are esters with alkylgroups of one to 4 carbon atoms, inclusive. Of those alkyl, methyl andethyl are especially preferred. When an alkanoate form is used, thediacetyl compounds are preferred. In Formulas VI, VII, VIII, and IX, itis contemplated that both R be hydrogen or that both R be alkanoyl.

When the compounds of Formulas II to IX are used for intravenousinjection or infusion, as mentioned above, sterile aqueous isotonicsolutions are preferred. For that purpose, it is preferred because ofincreased water solubility that a free acid or salt form he used. Forsubcutaneous or intramuscular injection, sterile solutions orsuspensions of the acid, salt, ester, alkanoate, or esteralkanoate formin aqueous or non-aqueous media are used. Tablets, capsules, and liquidpreparations such as syrups, elixers, and simple solutions, with theusual pharmaceutical carriers, are used for oral or sublingualadministration.

Prior to this invention, the only prostanoic acid derivatives isolatedfrom natural sources have been substances designated prostaglandins.See, for example, Horton, Experientia, 15, 113-114 (1965). Each of theknown prostaglandins is characterized by 0-110; and C-lSS hydroxy groupsand a 13,14-trans double bond. In addition, prostaglandin E (PGE has anoxo moiety at C-9; prostaglandin E (PGE has an oxo moiety at C-9 and a5,6-cis double bond; prostaglandin E (PGE has an oxo moiety at C-9,. and5,6-cis and 17,18-cis double bonds; prostaglandin F (PGF has a 0-90:hydroxy group; prostaglandin F (PG F Has a 0-90: hydroxy group and a5,6-cis double bond; prostaglandin F (PGF has a C-9a hydroxy group, and5,6-cis and 17,18-cis double bonds.

Other prostanic acid derivatives have been obtained by chemicaltransformations of the above-mentioned prostaglandins. For example,borohydride reduction of the C-9 oxo moiety of PGE results in theformation of PGF and the corresponding isomer with the C-9 hydroxy groupin the a configuration, PGF See, for example, Bergstrom et al., Acta.Chem. Scand., 16, 969-974 (1962).

Prior to this invention, prostanoic acid derivatives with oxygen atother than C-1, C-9, 0-11, and 0-15 had not been described. It has nowbeen discovered that certain prostanoic acid derivatives with hydroxygroups at 0-19 can be isolated from human seminal plasma by the novelprocess of this invention.

Earlier work with human seminal plasma resulted in the isolation of PGEPGE PGE PGF and PGF Samuelsson, J. Biol. Chem. 238, 3229-3234 (1963). Inthat work, it was discovered that reversed phase partitionchromatography on a hydrophobic diatomaceous earth (Super-Cel) supportwould separate individual PGE type compounds from each other, andindividual PGF type compounds from each other, but would not completelyseparate the PGE type compounds from the PGF type compounds. It was alsodiscovered in that work that partition chromatography on silicic acidwould separate PGE type mixtures from PGF type" mixtures but would notseparate the individual PGE type compounds from each other or theindividual PGF type compounds from each other.

Also in the earlier work of Samuelsson, ibid., use was made of earlierobservations that both PGE type and PGF type compound concentrations canbe estimated by measuring smooth muscle-stimulating activity on isolatedrabbit duodenum, the activity being expressed in PGE -intestinal units,one such unit corresponding to one microgram of a standard prepared fromcrystalline PGE That measurement does not, of course, distinguishbetween PGE type" and PGF type compounds.

In working with the PGE type compounds in human seminal plasma,Samuelsson, ibid., also used the earlier observation that treatment of aPGE type compound with sodium hydroxide solution causes formation of achromophore with an ultraviolet absorption maximum at 278 m PGF typecompounds do not form that chromophone on treatment with sodiumhydroxide. Therefore, that reaction can be used to estimate PGE typecompound concentrations but not PGF type compound concentrations.

The novel process of this invention differs in several substantialrespects from the process described by Samuelsson, ibid. Hereinafter,for convenience, the latter process will be referred to as theSamuelsson process to distinguish it from the novel process of thisinvention.

In the Samuelsson process, the total mixture of smoothmuscle-stimulating compounds was extracted from human seminal plasma,partitioned between petroleum ether and an ethanol-water solution, andthen subjected to partition chromatography on silicic acid, variousmixtures of ethyl acetate and benzene being used for elution, and thecontent of each eluate fraction being measured both by weight and bysmooth muscle-stimulating activity, i.e., in terms of PGE -intestinalunits. Next in the Samuelsson process, certain eluate fractionsrepresenting a peak of smooth muscle-stimulatory activity thought to bedue to various PGE-type compounds were combined and subjected to acombination of reversed phase partition chromatography, partitionchromatography, and thin layer chromatography, whereby PGE PGE and PGEwere separated, and isolated and identified as methyl esters.

Then, in the Samuelsson process, certain other eluate fractionsrepresenting another and slower moving peak of smooth muscle-stimulatingactivity thought to be caused by various PGF-type compounds werecombined and subjected to reversed phase partition chromatography, thecontent of each eluate fraction being measured by smoothmuscle-stimulatory activity. Certain eluate fractions from the latterchromatographic process, representing a peak of smoothmuscle-stimulating activity, were combined and evaporated to give aresidue which was treated with diazornethane to produce methyl esters ofthe various prostanoic acids in those fractions. That methyl estermixture Was then subjected to thin layer chromatography whereby themethyl ester of PGF and the methyl ester of PGF were separated,isolated, and identified.

In the novel process of this invention, the acidic fraction of a lipidextract of human seminal plasma is obtained by extraction andpreliminary purification, substantially as described by Samuelsson,ibid. That acidic fraction is then subjected to partition chromatographyon silicic acid, substantially as described by Samuelsson, ibid., exceptthat the content of each eluate fraction is measured by evaporating analiquot of each fraction to dryness, treating the residue with 0.5 Nsodium hydroxide in 50% aqueous ethanol, and, after minutes, measuringthe ultraviolet absorbancy at 278 me.

By plotting eluate fraction number against ultraviolet absorbancy at 278m four main peaks of absorbancy are observed in this novel process. Whena small amount of PGF containing tritium in place of hydrogen on the9-hydroxyl group is added to the mixture prior to the partitionchromatography, and the tritium activity of the various eluate fractionsmeasured, a single peak of tritium activity is observed. That tritiumactivity peak coincides roughly with the third peak (in order of ease ofelution from the chromatogram) of ultraviolet absorbancy.

Since it was observed by Samuelsson, ibid., that PGF- type compounds arepresent in human seminal plasma, and since partition chromatography onsilicic acid as described by Samuelsson, ibid., does not separate theindividual PGF-type compounds from each other, the third peak ofultraviolet absorbancy obtained by the novel process of this inventionmust contain all of the PGF-type compounds in the human seminal plasma.

As mentioned above, it is known that PGF-type compounds do not produce achromophore which absorbs at 278 me on treatment with dilute sodiumhydroxide solution. Therefore, it was completely unexpected andsurprising that eluate fractions expected to contain PGF-type compoundswould also contain compounds which give the 278 m chromophore ontreatment with dilute sodium hydroxide solution. Thus, an essential partof the novel process of this invention is the step of detectingunexpected compounds with substantially the same mobility as PGF-typecompounds during partition chromatography on silicic acid, by treatmentof aliquots of eluate fractions with alkali metal hydroxides, preferablysodium hydroxide or potassium hydroxide, and preferably in dilutesolution, and then measurement of the ultraviolet absorbancy of thetreated eluate at 278 111m.

The nature of the solvent is not critical as long as both alkali metalhydroxide and compounds to be treated are in solution together. Amixture of Water and a lower alkanol such as methanol or ethanol ispreferred. The eluate aliquot can be treated directly with the alkalimetal hydroxide or the aliquot can first be evaporated to dryness.

Further in the novel process of this invention, the eluates of the thirdultraviolet absorbancy peak, obtained by partition chromatography asdescribed above by the novel process of this invention and containingthe unexpected com ounds of this invention as well as PG-F-typecompounds, are combined and subjected to reversed phase chromatography.An essential aspect of that novel reversed phase chromatography step ofthe novel process of this invention is that the step provide anessentially complete separation of PGF and PGF from the unexpectedcompounds of this invention. Another important aspect of this reversedphase chromatography step is that it provide a substantial although notnecessarily complete separation of the unexpected compounds of thisinvention from each other.

The reversed phase partition chromatography which is part of the novelprocess of this invention is similar to that described by Norman et al.,J. Biol. Chem., 233, 8724585 (1958). It was discovered quiteunexpectedly, however, that use of a mixture of equal volumes ofchloroform and isooctanol as the stationary phase on hydro'pholickieselguhr (diatomite or diatomaceous earth), and a mixture of 114volumes of methanol and 186 volumes of water as the moving phase wouldpermit the compounds of this invention to pass through thechromatographic column substantially more rapidly than the PGF and PGFand thus be separated from the latter compounds. Moreover, by this novelchromatographic procedure, the four 19-hydroxyprostanic acids of thisinvention, i.e., the compounds of Formulas II, III, IV and V, quiteunexpectedly moved through the chromatographic column at substantiallydiiferent rates, thus effecting a substantial separation of those fourcompounds from each other.

The above-described novel reversed phase partition chromatographicseparation differs from the reversed phase chromatographic separationdescribed by Samuelsson, ibid., for the separation of PGF and PGF notonly in the result obtained but also in the means of obtaining theresult. A substantially smaller proportion of methanol is present in themoving phase in the novel process of this invention than was used in theSamuelsson process. The latter process used a moving phase containingvolumes of methanol and volumes of water. In the novel process of thisinvention, 114 volumes of methanol and 186 volumes of water is presentin the movingphase. In the novel process of this invention, the movingphase can contain methanol and water in volume-volume ratios rangingfrom about 110 volumes of methanol plus 190 volumes of water to about120 volumes of methanol plus volumes of water. When larger proportionsof methanol are used, PGF and PGF tend to remain with the compounds ofthis invention during chromatography. When smaller amounts of methanolare used, the separation of the individual compounds of this inventionis poorer than when a methanol-water mixture within the preferred rangeis used.

The above-described novel reversed phase chromatography process difiersfrom the reversed phase part of the Samuelsson process in anothersignificant manner. In the Samuelsson process, the content of thevarious eluate fractions from the reversed phase partitionchromatography was determined by measuring smooth muscle-stimulatoryactivity of those fractions. In this part of the novel process of thisinvention, the content of the various eluate fractions was measured bydetermining the ultraviolet absorbancy of each fraction at 278 m bothbefore and after treatment with a dilute alkali metal hydroxidesolution.

preferably sodium hydroxide or potassium hydroxide, as described abovefor the partition chromatography on silicic acid. Since the Samuelssonprocess had yielded only PGF-type compounds and since such compounds donot have a chromophore absorbing at 278 m nor yield such a chromophoreon treatment with dilute alkali, it was completely unexpected andsurprising that compounds could be detected in these eluate fractions inthat manner.

By the above-described novel reversed phase partition chromatographyprocess, a substantial separation of the unexpected compounds of thisinvention is obtained. The novel l9-hydroxyprostanoic acid of FormulaIII moves more rapidly on the chromatographic column than the otherl9-hydroxyprostanoic acids of Formulas II, IV, and V. Indeed, certain ofthe first group of eluate fractions obtained from the reversed phasechromatograms contain the Formula III l9-hydroxyprostanoic acidessentially free of Formula II, IV and V l9-hydroxyprostanoic acids.Hence, as will be described in greater detail hereinafter, thosefractions can be combined and the Formula III 19- hydroxyprostanoic acidisolated therefrom in essentially pure form by conventional techniques,for example, by evaporation of the solvents in the combined eluates. Ifdesired, the essentially pure evaporation residue can be crystallizedfrom a suitable solvent or mixture of solvents or subjected tochromatography, preferably preparative thin layer chromatography by themethod of Gren et al., J. Lipid Research 5, 117-120 (1964). Especiallypreferred as thin layer chromatography systems for that purpose aremixtures of ethyl acetate-aceticacid-methanol-2,2,4-trimethylpentane-water, especially 110: l: l 100 byvolume, and mixtures of ethyl acetate-aceticacid-2,2,4-trimethylpentane-water, especially l10:20:30:100 by volume.

The 19-hydroxyprostanoic acids of Formulas H and V are usually notcompletely separated by the above-described novel reversed phasepartition chromatographic process. When essentially pure samples ofthose compounds are desired, it is preferred to combine the variouseluate fractions which contain them and then isolate the mixture byconventional methods, for example, evaporation of the solvents in thecombined eluates. The mixture of the two l9-hydroxyprostanoic acids isthen separated by preparative thin layer chromatography as described byGren et al., ibid., preferably using silica gel containing a smallamount of silver nitrate, advantageously, about one part by weight ofsilver nitrate to about 25 parts of the silver gel, added as describedby Gren et al., ibid. Also, it is preferred to use a mixture of ethylacetateacetic acid-methanol-2,2,4-trimethylpentane-water, especially110: 10: :10:100 by volume as the solvent system. With that preferredprocedure, the Formula II 19-hydroxyprostanoic acid has an R value about0.68 and the Formula V l9-hydroxyprostanoic acid has an R value about0.48, thus indicating essentially complete separation. The essentiallypure Formula II and Formula V 19-hydroxyprostanoic acids can be isolatedfrom the thin layer chromatogram by conventional techniques, forexample, as described by Gren et al., ibid.

The novel 19-hydroxyprostanoic acid of Formula IV moves more slowly onthe reversed phase partition chromatographic column than any of theFormula II, III, and V l9-h'ydroxyprostanoic acids. Therefore a group ofthe last eluate fractions from the reversed phase partition chromatogramwill contain this substance in substantially pure form. However, smallamounts of Formula II and perhaps traces of Formula Vl9-hydroxyprostanoic acids may be present in those final eluates. Toobtain the Formula IV l9-hydroxyprostanoic acid in essentially pureform, the combined final eluate fractions are evaporated to dryness andthe residue subjected to preparative thin layer chromatography asdescribed by Gren et al., ibid., preferably using silica gel containinga small amount of silver nitrate as described above, and the specificethyl acetateacetic acid-methanol-2,2,4-trimethylpentane-water mixturedescribed above. The Formula IV l9-hydroxyprostanoic acid is therebyseparated from the other 19- hydroxyprostanoic acids and can be isolatedaccording to the Gren et al., ibid. The resulting essentially pureFormula IV 19-hydroxyprostanoic acid gives a single spot with R 0.36when subjected to thin layer chromatography silicic acid with a mixtureof ethyl acetate-acetic acid-2,2,4- trimethylpentane-water, 20 3 0: 100.

If it is desired to obtain only Formula IV and/or Formula Vl9-hydroxyprostanoic acids, i.e., only those compounds with C doublebonds, it is advantageous to treat the entire mixture of prostanoicacids in the third ultraviolet absorbancy peak of the above-describedsilicic acid partition chromatogram with dilute alkali metal hydroxidesolution, preferably substantially as described for the compounddetection method in the above-described novel reversed phase partitionchromatography except on an appropriately larger scale. That treatmentconverts the Formula II l9-hydroxyprostanoic acid to the Formula IV19-hydroxyprostanoic acid, and the Formula III 19-hydroxyprostanoic acidto the Formula V 19-hydroxyprostanoic acid. The reaction involved isisomerism of the Clem) double bond to the C position. It is the C802)double bond in conjugation with the 9-oxo group and the C double bondwhich is the chromophore that absorbs ultraviolet light at 27 8 m Theresulting mixture of Formula IV and Formula V l9-hydroxyprostanoic acidsis then readily separated by the combination of the novel reversed phasepartition chromatography and the novel preparative thin layerchromatography, each as described above. Essentially pure Formula IV andFormula V 19-hydroxyprostanoic acids are thereby obtained.

Alternatively, the essentially pure Formula II and Formula IIIl9-hydroxyprostanoic acids can individually be transformed toessentially pure Formula IV and Formula V 19-hydroxyprostanoic acids,respectively, by the abovedescribed treatment with dilute alkali metalhydroxide solution.

The 19-hydroxyprostanoic acids of this invention, i.e., compounds ofFormulas H, III, IV, and V, can be transformed into various types ofesters, for example, compounds of Formulas VI to IX wherein R is analkyl moiety and R is hydrogen, wherein R is an alkanoyl moiety and R ishydrogen or a pharmacologically acceptable cation, and wherein R is analkyl moiety and R is an alkanoyl moiety, all as defined above.

'Esterification of the carboxyl moiety in Formula II, III, IV, and V19-hydroxyprostanoic acids and in Formula VI, VII, VIII, and IXcompounds wherein R is hydrogen and R is hydrogen or alkanoyl can beaccomplished by interaction of the free acid with the appro' priatediazohydrocarbon. For example, when diazomethane is thus used, methylesters are produced. Similar use of diazoethane, diazobutane, andl-diazo-Z-ethyl-hexane, and the like gives ethyl, butyl, and2-ethylhexyl esters of the Formula II to IX acids.

Esterification with the diazohydrocarbons is carried out by mixing asolution of the diazohydrocarbon in a suitable inert solvent, preferablydiethyl ether, with the prostanoic acid reactant, advantageously in thesame or a different inert diluent. After the esterification reaction iscomplete, the solvent is removed by evaporation and the ester purifiedif desired by conventional methods, preferably by chromatography. It ispreferred that contact of the acid reactants with the diazohydrocarbonbe no longer than necessary to effect the desired esterification,preferably about one to about ten minutes, to avoid undesired molecularchanges. Diazohydrocarbons are known in the art or can be prepared bymethods known in the art. See, for example, Organic Reactions, JohnWiley and Sons, Inc., New York, N.Y., vol. 8, pp. 389-394 (1954).

An alternative method for esterification of the carboxyl moiety ofFormula II to IX l9-hydroxyprostanoic acids comprises transformation ofthe free acid to the corresponding silver salt, followed by interactionof that salt with an alkyl iodide. Examples of suitable iodides aremethyl iodide, ethyl iodide, butyl iodide, isobutyl iodide, tert-butyliodide, and the like. The silver salts are prepared by conventionalmethods, for example, by dissolving the acid in cold dilute aqueousammonia, evaporating the excess ammonia at reduced pressure, and thenadding the stoichiometric amount of silver nitrate.

Carboxyacylation of the two hydroxy groups in the Formula II, III, IV,and V 19-hydroxyprostanoic acids and in the Formula VI, VII, VIII, andIX compounds wherein R is hydrogen or alkyl and R is hydrogen isaccomplished by interaction of the hydroxy compound with acarboxyacylating agent, preferably the anhydrides of alkanoic acids. Forexample, use of acetic anhydride gives the corresponding diacetate.Similar use of propionic anhydride, butyric anhydride, and isobutyricanhydride, gives the corresponding dicarboxyacylates.

The carboxyacylation is advantageously carried out by mixing the hydroxycompound and the acid anhydride preferably in the presence of a tertiaryamine such as pyridine or triethylamine. A substantial excess of the anhydride should be used, preferably about 10 to about 10,000 moles ofanhydride per mole of the hydroxy compound reactant. The excessanhydride serves as a reaction diluent and solvent. An inert organicdiluent, for example, dioxane, can be added. It is preferred to useenough of the tertiary amine to neutralize the carboxylic acid producedby the reaction as well as any free carboxyl groups present in thehydroxy compound reactant.

The carboxyacylation reaction is preferably carried out in the rangeabout to about 100 C. The necessary reaction time will depend on suchfactors as the reaction temperature and the nature of the anhydride andtertiary amine reactants. With acetic anhydride, pyridine, and a 25 C.reaction temperature, a 12 to 24-hour reaction time should be used.

The carboxyacylated product is isolated from the reaction mixture byconventional methods. For example, the excess anhydride can bedecomposed with water, and the resulting mixture acidified and thenextracted with a solvent such as diethyl ether. The desiredcarboxyacylate will usually be extracted by the ether and is recoverabletherefrom by evaporation. If desired, the carboxyacylate can be purifiedby conventional methods, preferably by chromatography.

Each of the above-described l9-hydroxyprostanoic acids, i.e., FormulasII, III, IV, and V, and Formulas VI, VII, VIII, and IX wherein R ishydrogen are transformed to pharmacologically acceptable salts byneutralization with appropriate amounts of the corresponding inorganicor organic base, examples of which correspond to the cations listedabove. These transformations can be carried out by a variety ofprocedures known in the art to be generally useful for the preparationof inorganic, i.e., metal or ammonium, salts, amine acid addition salts,and quaternary ammonium salts. The choice of procedure will depend inpart upon the solubility characteristics of the particular salt to beprepared. In the case of the inorganic salts, it is usually suitable todissolve the 19-hydroxyprostanoic acid in water containing thestoichiometric amount of a hydroxide, carbonate, or bicarbonatecorresponding to the inorganic salt desired. For example, such use ofsodium hydroxide, sodium carbonate, or sodium bicarbonate gives asolution of the sodium salt of the Formula II to IX 19-hydroxyprostanoicacid. Evaporation of the water or addition of a watermiscible solvent ofmoderate polarity, for example, a lower alkanol or a lower alkanonegives the solid inor ganic salt if that form is desired.

To produce an amine salt, the 19-hydroxyprostanoic acid can be dissolvedin a suitable solvent of either moderate or low polarity. Examples ofthe former are ethanol, acetone, and ethyl acetate. Examples of thelatter are diethyl ether and benzene. At least a stoichiometric amountof the amine corresponding to the desired cation is then added to thatsolution. If the resulting salt does not precipitate, it can usually beobtained in solid form by addition of a miscible diluent of loW polarityor by evaporation. If the amine is relatively volatile, any excess caneasily be removed by evaporation. It is preferred to use stoichiometricamounts of the less volatile amines.

Salts wherein the cation is quaternary ammonium are produced by mixingthe 19-hydroxyprostanoic acid with the stoichiometric amount of thecorresponding quaternary ammonium hydroxide in water solution, followedby evaporation of the water.

As discussed above, the novel Formula II, III, IV, and V prostanoic acidderivatives of this invention are obtained from human seminal plasma,along with numerous other prostanoic acid derivatives, for example, PGEPGE and PGE These other prostanoic acid derivatives have differentbiological and pharmacological properties than the novel compounds ofthis invention. In order to be useful for the intended pharmacologicalpurposes set forth hereinabove, each of the novel Formula II, III, IV,and V compounds must be essentially free of these other prostanoicacids. Moreover, human seminal plasma is known to contain many othersubstances, for example, proteins, enzymes, cellular material, andnumerous other organic acids or salts thereof. The novel Formula II,III, IV, and V compounds of this invention must also be essentially freeof these other human seminal plasma constituents which would alsointerfere with the intended uses of these novel compounds. By the termessentially free here is meant a degree of freedom from these impuritiessuch that these novel compounds are suitable for their intendedpharmacological uses, including parenteral administration to humans.When these novel compounds are purified to this degree, they areconsidered to be essentially pure and useable for all of theabove-described pharmacological purposes.

The invention can be more fully understood by the following examples.

EXAMPLE l.ISOLATION OF l9-HYDROXY- PROSTANOIC ACIDS FROM HUMAN SEMI- NALPLASMA (A) Extraction and preliminary purification Samples of humanseminal plasma are obtained from the source described by Samuelsson,i-bid., and are added to ethanol and stored at 20 C. until processed.

A 650-ml. portion of plasma in 2000 ml. of ethanol is centrifuged atabout 25 C., and the clear supernatant is decanted. The sediment isresuspended in 2500 ml. of ethanol and the mixture is centrifuged. Thecombined supernatants are filtered, and evaporated to 200 ml. at 10 mm.pressure and 40 C. The resulting solution is then mixed with 200 ml. ofwater, and the mixture is acidified to pH 3 with hydrochloric acid.

The acidified solution is extracted three times with diethyl ether. Thediethyl ether extracts are combined and Washed with successive portionsof Water until further water washings are neutral. The Washed diethylether extracts are then evaporated to dryness under reduced pressure,and the residue is subjected to a three-stage distribution betweenpetroleum ether and ethanol-water (2:1). The combined aqueous ethanolphases are evaporated to a small volume, and then diluted with water,acidified, and extracted three times with diethyl ether. The combineddiethyl ether extracts are evaporated to give 541 mg. of residue.

(B) Partition chromatography on silicic acid The residue (541 mg.) fromPart A is mixed with 25 micrograms of PGF containing tritium in place ofhydrogen in the 9-hydroxy group. (Samuelsson, Biochem. Biophys, Acta 84,709 (1964)). The mixture is then subjected to partition chromatographyon 25 g. of silicic acid, eluting with benzene containing increasingconcentrations of ethyl acetate, as described by Samuelsson, ibid. Eacheluate fraction is 42 ml. Fractions 1 to 6 are eluted with ethylacetate-benzene 3:7 (v.:v.). Fractions 7 to 27 are eluted with ethylacetate-benzene 6:4 (v.:v.). Fractions 28 to 39 are eluted with ethylacetate-benzene 8:2 (v.:v.). A small aliquot of each fraction isevaporated to dryness, and the residue is mixed with 0.5 N sodiumhydroxide in 50% aqueous ethanol and allowed to stand at about 25 C. for30 minutes. The ultraviolet absorbancy at 278 m is then measured. Fourmain peaks of ultraviolet absorbancy are observed among the successiveeluate fractions.

The radioactivity of each eluate fraction, and hence the PGF;;., contentof each, is measured with a gas flow counter (Frieseke-Hoepfner FH 51)in the proportional range. The radioactive fractions coincide roughlywith the third main peak of ultraviolet absorbancy.

(C) Reversed phase partition chromatography The eluate fractionsrepresenting the third peak of ultraviolet absorbancy are combined andevaporated to dryness to give 74 mg. of residue. That residue issubjected to reversed phase chromatography according to the proceduresdescribed by Norman et al., J. Biol. Chem. 233, 872 (1958). See alsoreferences cited in Norman et al. An 18-gram column is used. The movingphase of the solvent system is methanol-water 114:186- (v.:v.). Thestationary phase of the solvent system is chloroformisooctanol 15:15(v.:v.). The ultraviolet absorbancy of an aliquot of each eluatefraction is measured at 278 m before and after treatment with sodiumhydroxide solution as described in Part B, above.

FIG. 1 shows the results obtained in the above-described reversed phasepartition chromatography. The solid line represents absorbancy at 27 8 mfor the various eluate fractions after treatment with sodium hydroxidesolution. The dotted line with solid points represents absorbancy of thevarious eluate fractions before treatment with sodium hydroxidesolution. The dashed line with open points represents the calculateddifference in absorbancy between the solid line and the broken line withsolid points. Thus each point on the broken line with open pointsrepresents an absorbancy increase due to treatment of an eluate fractionaliquot with sodium hydroxide solution, and hence is a measure of theamount of material in that fraction which does not absorb at 278 mbefore treatment with sodium hydroxide but which does absorb after thattreatment.

(D) Thin layer chromatography and characterization Thin layerchromatography described below is carried out according to theprocedures described by Gren et al., J. Lipid Research 5, 117-120(1964). Two solvent systems are used. System A consists of ethylacetateacetic acid-methanol-2,2,4 trimethylpentane-water, 110::15:10:100 by volume. System B consists of ethyl acetate-aceticacid-2,2,4-trimethylpentane-water, 110:20: 302100 by volume. System A isused with a mixture of silica gel and silver nitrate, 25:1 by Weight.System B is used with silica gel. In both cases, the specific silica geland the preparation and use of the plates are as described by Gren etal., ibid.

(a) Fractions 28 to 35 (see FIG. 1) from the abovedescribed reversedphase partition chromatography are combined and evaporated to dryness togive 10 mg. of essentially pure 15,19-dihydroxy-9-oxoprosta-cis-5,10-trans-lS-trienoic acid.

U.V. (ethanol) 217 m (e=10,000). I.R. (principal bands; chloroformsolution) 5.86, 6.31,

103a. Thin layer chromatography:

A single spot with R 0.52 using solvent system A. A single spot with R0.38 using solvent system B.

(b) Fractions 40 to 52 (see FIG. 1) from the abovedescribed reversedphase partition chromatography are combined and evaporated to dryness togive 19 mg. of

14 residue. Preparative thin layer chromatography according to Gren etal., ibid., using solvent system A separates that residue into twoessentially pure compounds, one with R 0.68 and one with R 0.48.

The compound with R 0.68 is 15,19-dihydroxy-9-oxoprosta-10,traus-13-dienoic acid.

U.V. (ethanol) 217 my. (e=l1,000).

I.R. (principal bands; chloroform solution) 5.86, 6.31,

Thin layer chromatography: A single spot with R 0.38

using solvent system B.

The compound with R 0.48 is 15,19-dihydroxy-9-oxoprosta-cis-S ,8 12),trans-13-trienoic acid.

U.V. (ethanol) 278 m (e=20,000).

LR. (principal bands; chloroform solution) 5.92, 6.09,

Thin layer chromatography: A single spot with R;- 0.37

using solvent system B.

(c) Fractions 54 to 61 (see FIG. 1) from the abovedescribed reversedphase partition chromatography are combined and evaporated to dryness togive a residue. Preparative thin layer chromatography according to Grenet al., ibid., using solvent system A gives a spot from whichessentially pure 15,19-dihydroxy-9-oxoprosta- 8(12),trans-l3-dienoicacid is isolated.

U.V. (ethanol) 278 m (e=21,000)- I.R. (principal bands; chloroformsolution) 5.92, 6.09,

Thin layer chromatography: A single spot with R 0.36

using solvent system B.

EXAMPLE 2.-ISOLATION OF 8( 12)-UNSATU- RATED 19 HYDROXYPROSTANOIC ACIDSFROM HUMAN SEMINAL PLASMA Following the procedure of Example 1, Parts Aand B, the acidic fraction of a lipid extract of a batch of humanseminal plasma is purified and subjected to partition chromatography onsilicic acid. The eluate fractions containing the third main peak ofultraviolet absorbancy at 278 m are combined and evaporated to drynessto give 24 mg. of residue. That residue is then mixed with 0.5 N sodiumhydroxide in 50% aqueous ethanol, and the mixture is heated for 45minutes at 40 C. The resulting solution is then acidified and extractedrepeatedly with diethyl ether. The diethyl ether extracts are combinedand evaporated to dryness to give a residue which is subjected toreversed phase chromatography, following the procedure of Example 1,Part C. Only two main peaks of ultraviolet absorbancy appear. The eluatefrac tions containing the first peak of ultraviolet absorbancy arecombined and evaporated to give 10.0 mg. of essentially pure15,19-dihydroxy-9-oxoprosta-cis-5,8( 12) ,trans-13- trienoic acid; thinlayer chromatography as in Example 1, Part D, gives a single spot withthe same R given in Example 1.

The eluate fractions containing the second peak of ultravioletabsorbancy are combined and evaporated to dryness to give 11.0 mg. ofessentially pure 15,19-dihydroxy-9-oxoprosta-8(12),trans-13 dienoicacid; thin layer chromatography as in Example 1, Part D, gives a singlespot with the same R given in Example 1.

EXAMPLE 3.CONVERSION OF 10(l1)-UNSATU- RATED 19-HYDROXYPROSTANOIC ACIDSTO 8(12) UNSATURATED 19 HYDROXYPROS- TANOIC ACIDS Following theprocedure described in Example 2 for the treatment of thel9-hydroxyprostanoic acid mixture with sodium hydroxide solution,essentially pure 15,19- dihydroxy-9-oxoprosta 10,trans-13-dienoic acidand essentially pure 15,19-dihydroxy-9-oxoprosta-cis-5,l0-trans-l3-trienoic acid are separately transformed by sodium hydroxidetreatment to essentially pure 15,19-di- 15 hydroxy-9-oxoprosta8(l2),trans-13-dienoic acid and essentially pure 1519-dihydroxy-9oxoprosta-cis5,8 1 2) trans-13-trienoic acid,respectively.

EXAMPLE 4.-METHYL 15 l9-DIHYDROXY-9- OXOPROSTA-l0,TRANS-13-DIENOATEEssentially pure 15,l9-dihydroxy-9-oxoprosta-l0,transl3-dienoic acid (2mg.) is dissolved in a mixture of methanol and diethyl ether 1:9. Adiethyl ether solution of diazomethane (about 200 mg.) is added, and themixture allowed to stand at about 25 C. for 5 minutes. The reactionmixture is then evaporated to dryness to give methyl15,19-dihydroxy-9-oxoprosta-10,trans-13-dienoate.

I.R. (principal bands; chloroform solution) 5.86, 6.31,

Mass spectrum: 279, 299, 317, 348, 366 m/e on apparatus of Ryhage,Ar-kiv Kemi, 16, 19 (1960).

EXAMPLE 5 .--METHYL 15 1 Q-DIHYD ROXY-9- OXOPROSTA-CIS-S l 0,TRANS- lB-TRIENOATE Following the procedure of Example 4, essentially pure15,19-dihydroxy 9-oxoprosta-cis 5,10,trans-13-trienoic acid istransformed to methyl15,19-dihydroxy-9-oxoprosta-cis-5,10,trans-l3-trienoate.

LR. (principal bands; chloroform solution) 5.86, 6.31,

Mass spectrum: 315, 328, 346, 364 m/e on apparatus of Ryhage, ibid.

N.M.R.: Multiplets at 5.25-5.50 and 5.50-5.75 ppm. (5); doublet at 1.15ppm. (6); singlet at 1.25 ppm. (5). Spectrum taken with a Varian A-6Ospectrophotometer on deuterochloroform solution with tetrarnethylsilaneas internal standard.

EXAMPLE 6.METHYL 1 5,19DIHYDROXY9 OXOPROSTA-8 12 ,TRANS- 1 B-DIENOATEFollowing the procedure of Example 4, essentially pure 15,19-dihydroxy 9oxoprosta-8( l2),trans 13 dienoic acid is transformed to methyl15,19-dihydroxy-9-oxoprosta-8( l2 ,trans-13-dienoate.

LR. (principal bands; chloroform solution) 5.92, 6.09,

EXAMPLE 7.METHYL 15,19-DIHYDROXY-9-OXO- PROSTA-CIS-S ,8 12 ,TRANS- 1 3-TRIEN OATE Following the procedure of Example 4, essentially pure15,19- dihydroxy 9 oxoprostacis-5,8(12),trans-l3-trienoic acid istransformed to methyl 15,19-dihydroxy-9- oxoprosta-cis-5,8 12) ,trans-13-trienoate.

LR. (principal bands; chloroform solution) 5.92, 6.09,

EXAMPLE 8.-METHYL 15 1 9-DIACETOXY-9- OXOPROSTA-10,TRANS-l 3-D1ENOATEMethyl 15,19-dihydroxy-9-oxoprosta-10,trans-13-dienoate (2 mg.) is mixedwith acetic anhydride (0.5 ml.) and pyridine (0.5 ml.). The resultingmixture is allowed to stand at 25 C. for 18 hours. The reaction mixtureis then cooled with ice, diluted with water, and acidified with dilutehydrochloric acid to pH 1. That mixture is then extracted three timeswith diethyl ether. The diethyl ether extracts are combined and washedsuccessively with 2 N hydrochloric acid, 1 N sodium bicarbonatesolution,

and water. The ether is then evaporated under reduced pressure to givemethyl l5,l9-diacetoxy-9-oxoprosta-10, trans-13-dienoate. On oxidativeozonolysis followed by reaction with diazomethane, that compound givesmethyl 2,6-diacetoxyheptanoate and trimethyl octane-l,2,8tricarboxylate.

Following the procedure of Example 8, methyl 15,19-dihydroxy-9-oxoprosta-8(12),trans 13 dienoate, methyl 15,19-dihydroxy9-oxoprosta-cis-5,10,trans-1B-trienoate, and methyl 15,19-dihydroxy 9oxoprosta-cis-5,8(l2), trans-13-trienoate are transformed separately tomethyl 15,19 diacetoxy 9 oxoprosta-8(12),trans-l3-dienoate, methyll5,19-diacetoxy 9 oxoprosta-cis-5,l0,trans-l3- trienoate, and methyl15,19-diacetoxy-9-oxoprosta-cis- 5,8(l2),trans-l3-trienoate,respectively. On oxidative ozonolysis followed by reaction withdiazomethane, the methyl 15,19-diacetoxy 9 oxoprosta-cis-S,l0,trans-13-trienoate gives dimethyl glutarate, trimethyl tricarballylate, andmethyl 2,6-diacetoxyheptanoate.

Also following the procedure of Example 8 but substituting for theacetic anhydride, propionic anhydride and butyric anhydride, there areobtained the corresponding 15,19-dicarboxyacyl derivatives of methyl15,19-dihydroxy-9-oxoprosta-l0,trans-l3-dienoate. In a similar manner,the same 15,19-dicarboxyacyl derivatives of methyl 15,19 dihydroxy 9oxoprosta-8(12),trans-13- dienoate, methyl 15,19-dihydroxy 9oxoprosta-cis-5,10, trans-13-trienoate, and methyl15,19-dihydroxy-9-oxoprosta-cis-5,8(12),trans-13-trienoate is prepared.In a similar manner, each of the other esters of the novel 19-hydroxyprostanoic acids of this invention mentioned above is reactedwith acetic anhydride or with each of the other above-mentionedanhydrides to produce the corresponding 15,19-dicarboxyacyl derivatives.

Also following the procedure of Example 8, essentially pure15,19-dihydroxy-9-oxoprosta-10,trans 13 dienoic acid, 15,19-dihydroxy 9oxoprosta-8(12),trans-13-dienoic acid,15,l9-dihydroxy-9-oxoprosta-cis-5,10,trans-13- trienoic acid, and15,19-dihydroxy-9-oxoprosta-cis-5,8- (12),trans-13-trienoic acid areeach separately reacted with acetic anhydride or with each of the otherabovementioned anhydrides to produce the corresponding 15,l9-dicarboxyacyl derivatives. In the latter cases, the sodiumbicarbonate wash is omitted. Then, following the procedure of Example 4,each of those 15,19-diacyloxy dienoic and trienoic acids is separatelyreacted with di azomethane or with each of the other above-mentioneddiazohydrocarbons to produce the same esterified dicarboxyacylderivatives produced by the alternative route, i.e., firstesterification and then carboxyacylation.

EXAMPLE 9.-SODIUM l5, 19-DIHYDROXY-9- OXO PROSTA- 10,TRANS- l3-DIENOATEEssentially pure 15 19-dihydroxy-9-oxoprosta-10,trans- 13-dienoic acid(2 mg.) is dissolved in 3 ml. of waterethanol 1:1. The solution iscooled to about 10 C. and is neutralized with an equivalent amount of0.1 N aqueous sodium hydroxide solution. Evaporation to dryness givesessentially pure sodium15,19-dihydroxy-9-oxoprosta-10,trans-13-dienoate.

Following the procedure of Example 15 but using in place of theessentially pure 15,19-dihydroxy-9-oxoprosta- 10,trans-13-dienoic acid,essentially pure 15,19-dihydroxy- 9-oxoprosta-cis-5,10,trans 13 trienoicacid; essentially pure 15,19 dihydroxy 9oxoprosta-8(l2),trans-13-dienoic acid; essentially pure15,19-dihydroxy-9-oxoprostacis-5,8( 12) ,trans-l 3-trienoic acid;15,19-dihydroxy-9-oxoprostanoic acid; and15,19-diacetoxy-9-oxoprostanoic acid, there are obtained thecorresponding sodium salts.

Also following the procedure of Example 15 but using potassiumhydroxide, calcium hydroxide, tetramethylammonium hydroxide, andbenzyltrimethylammonium hydroxide, there are obtained the correspondingsalts of essentially pure l5,19-dihydroxy-9-oxoprosta-10,trans-13-dienoic acid.

1 7 What is claimed is: 1. A compound of the formula:

wherein R is hydrogen, alkyl of one to 8 carbon atoms, inclusive, or apharmacologically acceptable cation, and R is hydrogen or alkanoyl ofone to 8 carbon atoms, inclusive, provided that when R, is hydrogen, Ris alkyl of one to 8 carbon atoms, inclusive.

2. A compound according to claim 1 wherein R is hydrogen and R isalkanoyl of one to 8 carbon atoms, inclusive.

3. A compound according to claim 2 wherein R is acetyl.

4. A compound according to claim 1 wherein R is alkyl of one to 8 carbonatoms, inclusive, and R is hydrogen.

5. A compound according to claim 4 wherein R is methyl.

6. A compound according to claim 4 wherein R is ethyl.

7. A compound according to claim 1 wherein R is alkyl of one to 8 carbonatoms, inclusive, and R alkanoyl of one to 8 carbon atoms, inclusive.

8. A compound according to claim 7 wherein R is acetyl.

9. A compound according to claim 7 wherein R is methyl.

10. A compound according to claim 7 wherein R is methyl and R is acetyl.

References Cited Hamberg et al., Biochim. Biophys. Acta, 106, 215 1965).

LORRAINE A. WEINBERGER, Primary Examiner R. GERSTL, Assistant ExaminerU.S. Cl. X.R.

260211 R, 247.2 R, 268 R, 293.65, 326.3, 410, 429.9, 439 R, 448 R, 488R, 501.1, 501.15, 501.17, 501.2, 514 D; 424--305, 311, 312, 317

